Composition Comprising PIC For Treatment Of Cancer

ABSTRACT

The present disclosure relates to a composition comprising PIC for treatment of cancer. More particularly, the present disclosure discloses a composition for treatment of cancer comprising polyinosinic-polycytidylic acid, an antibiotic or polyamine compound, a positive ion, and optionally a virus, and the use thereof in manufacture of a medicament for treatment of cancer. No figure for publication

FIELD OF THE INVENTION

This application relates to medical field, especially treatment ofcancer. More particularly, this application relates to a pharmaceuticalcomposition for treatment of cancer comprisingpolyinosinic-polycytidylic acid (PIC), antibiotic or polyamine compound,positive ion, and optionally an inactivated or attenuated virus; and theuse thereof for treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer commonly refers to a malignant tumor. Under the influence ofcarcinogenic factors, some cells in tissues lose their normal growthregulation at the genetic level, leading to abnormal proliferation anddifferentiation, eventually becoming a tumor. Compared to a benigntumor, a malignant tumor grows faster, is invasive, prone tohemorrhagging, necrosis, ulceration, and often metastasis.

There are over 100 different known cancers that affect humans. The top 5common sites of cancer are in the lung, prostate, colorectum, stomach,and liver cancer for men, and in the breast, colorectum, lung, cervix,and stomach cancer for women. Jie He and Wanqing Chen et al havepublished a paper named Cancer Statistics in China, 2015 in CancerJournal for Clinicians. This research analysed and predicted theincidence and mortality rate of malignant tumor in China in the year of2015. Data showed that China expected 4,292,000 new cases of malignanttumor in 2015. The five most common cancers are lung and bronchialcancer, stomach cancer, liver cancer, esophageal cancer and colorectalcancer for men, and breast cancer, lung cancer, stomach cancer,colorectal cancer and esophageal cancer for women. Lung cancer is one ofthe most common malignant tumors in China, being the highest malignanttumors mortality rate.

According to the World Health Organization (WHO), there areapproximately 14 million new cancer cases and 8.2 million people diefrom cancer each year, which is an estimated 13% of all deathsworldwide. Cancer is the leading cause of death in both developed anddeveloping countries. The burden is expected to grow worldwide withestimated new cancer cases rising by about 70% over the next two decades(WHO). Therefore, the increasing trend of cancer incidence has forcedhumankind to work more on the cancer prevention and treatments.

Currently the primary methods to treat cancer includes surgicaltreatment, chemotherapy which uses cancer cells killing drugs, radiationtherapy which uses high energy radiation to kill cancer cells, targetedtherapy which uses substances targeting specific cancer cells to delivertargeted therapy, and immunotherapy which uses immune system to treatcancer.

In addition, the application of virus in cancer treatment has graduallybeen noticed. In 1912, DePace discovered cervical tumor regression afterinoculation of rabies vaccine to a female patient who was bitten by dog.Since then, there has been several reports on the use of virus for thetreatment of cancer. Especially between 1950 to 1960, virus treatment ofcancer underwent rapid development. In the 1970s, the development ofvirus treatment slowed down owing to the concern of virus pathogenicity,but the pace was picked up again in 1980s.

Guoqian Kuang et al described the potential mechanism of anticancerviruses, as well as the selection and method in clinical application(Current Status and Prospect of Cancer Virotherapy Clinical Studies,Journal of Guangxi Medical University, 1995 Vol. 12: 617-619).Currently, it is understood that the mechanism of anticancer virusesinvolves direct oncolytic action, enhancing immune system andstimulating release of cytokines. It is generally believed that thevirus used in cancer treatment should be non-tumorigenic and with goodantigenicity. Viruses reported for anticancer treatment includes: NDV,Mumps virus, Vaccinia virus, Sendai virus, HSV and Parvovirus (Kenney Set al., Viruses as oncolytic agents: a new age for “therapeutic”viruses. J Nati Cancer Inst, 1994, 86:1185). Lorence et al found inanimal studies that live virus showed significantly better anti-cancereffect compared to inactivated virus (Complete regression of humanneuroblastoma xenografts in athymic mice after local Newcastle diseasevirus therapy. J Nati Cancer Inst, 1994, 86:1228).

Some viruses can specifically kill tumor cells in cancer treatment, andare known as oncolytic virus. Oncolytic viruses can selectivelyreplicate inside cancer cells, resulting in cytopathic effects andimmune responses that leads to the death of tumor cells, while showingminimum effects on normal cells and tissues (Jiang Zhong, OncolyticVirus and Tumor Treatment, Foreign Medicine (Microbiology Section), 2004Vol. 27 Iss. No. 6).

At present, there is a significant number of reports on oncolyticviruses in the treatment of cancer. For example, WO2009/016433 describesusing recombinant non-VSV rhabdovirus (such as Maraba virus, Carajasvirus, Muir Springs virus, and/or Bahia grande virus) to curehyperproliferative disease (e.g. cancer). US2010/0297072A1 discloses acomposition for the treatment of cancer comprising oncolytic virus(paramyxovirus, reovirus, herpesvirus, adenovirus, and Semliki Forestvirus) and an immunostimulant (CTLA-4 blocking agent, IL-21, anti-CD40or granulocyte-macrophage colony stimulating factor (GM-CSF)), andadministering this composition in MCA205 fibrosarcoma or B16 melanomatumor-bearing mice significantly inhibited tumor growth.

Rabies virus belongs to the lyssavirus genus of rhabdoviridae family.Rabies virus has a distinct “bullet” shaped, helically symmetricnucleocapsid with an envelope, and contains single stranded RNA inside.Rabies virus is the pathogen for rabies disease. Anti-rabiesimmunization is one of the successful early vaccination examples. In the1880s, Pasteur pioneered the use of vaccine. Early rabies vaccines werenerve tissue-derived, subsequently embryonated egg-cultured vaccines,cell culture vaccines, and subunit vaccines were developed. Geneticallyengineered vaccines are under development (Shounan Tan, Fengyu Zhang,Studies on Rabies and Human Rabies Vaccine. Medical Information 2011Vol. 24:2841-2842). At present, the majority of rabies vaccines forhuman use are produced by inoculation of fixed virus strain (e.g.,CTN-1V strain, aG strain) in Vero cells, culturing to obtain the virusin a liquid, followed by inactivation, concentration and purification,and further freeze-drying after adding appropriate an amount of gelatinor sucrose as a protective agent (Yuhui Zhang, The Establishment ofRabies Vaccine Purification Technology. Chinese Journal of Biologicals1999 Vol. 12 Iss. No. 4: 231-232).

Jieguang Sun et al discovered that, by using rabies vaccine for humanuse as the sole active ingredient in the dosage of 2.5 IU to 10 IU dailythrough intramuscular injection route, the adverse effects of malignanttumor treatment were improved in an animal tumor model, and this alsoenhances macrophage phagocytosis (see CN100341571C). RU2414238C2 alsoreports a method of increasing cancer resistance, in which rabiesvaccine increases the antitumor resistance of the organism to9,10-dimethyl-1,2 benzanthracene.

Viruses have great potential in cancer treatment. Safer and moreeffective virus based anticancer drugs are needed in this field.

SUMMARY OF THE INVENTION

The present disclosure provides a composition for use in treatment ofcancer, comprising or consisting of: a) polyinosinic-polycytidylic acid(PIC), b) at least one antibiotic or at least one polyamine compound,and c) at least one positive ion.

In another aspect, the present disclosure provides a composition for usein treatment of cancer, comprising or consisting of: a)polyriboinosinic—polyribocytidylic acid (PIC), b) at least oneantibiotic or at least one polyamine compound, c) at least one positiveion, and d) optionally a virus.

In one embodiment, the composition comprises a)polyriboinosinic—polyribocytidylic acid (PIC), b) at least oneantibiotic or at least one polyamine compound, c) at least one positiveion, and d) a virus.

In some embodiments, the virus is inactivated, attenuated, or incapableof replication in a human subject. In some particular embodiments, thevirus is inactivated.

In some embodiments, the virus is selected from the group consisting ofrhabdoviridae, adeniviridae, arena viridae, astroviridae, bunyaviridae,cliciviridae, flaviviridae, hepatitis delta virus, hepeviridae,mononegavirales, nidovirales, piconaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae,retroviridae, and togaviridae. In some particular embodiments, the virusbelongs to Lyssavirus genus of rhabdoviridae family. In some particularembodiments, the virus is a rabies virus.

In some embodiments, the rabies virus is inactivated crude virus (e.g.,hamster kidney cells inactivated crude rabies antigen (HKC-ICRA)), orinactivated purified virus (e.g., hamster kidney cell inactivatedpurified rabies antigen (HKC-IPRA)).

In some embodiments, the rabies virus suitable for use in the presentdisclosure is rabies vaccine including, but not limited to, inactivated,subunit, genetically modified, and polypeptide vaccine. In particular,the rabies virus vaccine suitable for use in the present disclosure ishuman diploid cell vaccine (HDCV), or hamster kidney cell inactivatedpurified rabies vaccine (HKC-IPRV), or hamster kidney cell inactivatedcrude rabies vaccine (HKC-ICRV), or purified Vero cell rabies vaccine(PVRV), or purified chicken embryo cell (PCEC) rabies vaccine, orpurified duck embryo rabies vaccine (PDEV).

In some embodiments, the PIC are heterogeneous for molecular weight,where the molecular weight is equal to or greater than 66,000 Daltons.The value of 66,000 Daltons corresponds to the molecular size of 6.4sedimentation unit (Svedbergs). In some embodiments, the molecularweight of PIC is from 66,000 to 1,200,000 Daltons (equivalent to 6.4 to24.0 sedimentation unit). In some other embodiments, the molecularweight of PIC is equal to or greater than 150,000 Daltons. In some otherembodiments, the molecular weight of PIC is from 100,000 to 200,000Daltons, or from 300,000 to 4,000,000 Daltons, or from 500,000 to1,000,000 Daltons, or from 1,000,000 to 1,500,000 Daltons, or from1,500,000 to 2,000,000 Daltons, or from 2,000,000 to 2,500,000 Daltons,or from 2,500,000 to 3,000,000 Daltons, or from 3,000,000 to 3,500,000Daltons, or from 3,500,000 to 4,000,000 Daltons, or from 4,000,000 to4,500,000 Daltons, or from 4,500,000 to 5,000,000 Daltons.

In some particular embodiments, the antibiotic is selected from thegroup consisting of tacrolamycin, anthracycline, butyrin sulphate,gentamicin, hygromycin, amikacin, dideoxy kanamycin, nebramycin,β-lactam, neomycin, puromycin, streptomycin, streptozocin, and anycombination thereof. The polyamine compound is selected from the groupconsisting of arginine salt, spermidine, N-(3-aminopropyl),N-(3-aminopropyl)-1,4-butanediamine, spermine,OS-dimethylaminothiophosphate, poly-lysine, aminoglycoside, and anycombination thereof.

In some particular embodiments, the antibiotic is kanamycin. In someembodiments, the concentration of the antibiotic in the composition isfrom 10 unit/ml to 100,000 unit/ml, preferably from 100 unit/ml to10,000 unit/ml, more preferably from 500 unit/ml to 5,000 unit/ml.

In some embodiments, the positive ion is a cation and is selected fromthe group consisting of calcium, cadmium, lithium, magnesium, cerium,cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, andany combination thereof. In some particular embodiments, the positiveion is calcium. The positive ion may be in the form of any suitable saltor organic complex including, but not limited to, chloride, fluoride,hydroxide, phosphate or sulfate. For example, when the positive ion iscalcium, the calcium ion may be in the form of calcium carbonate,calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphateor calcium sulfate. In some embodiments, the concentration of thepositive ion in the composition is from 0.01 μmol to 10 mmol/ml,preferably from 0.02 μmol to 5 mmol/ml, more preferably from 0.1 μmol to1 mmol/ml, most preferably from 0.1 μmol to 100 μmol/ml.

In some embodiments, the ratio of the virus to PIC is selected from thegroup consisting of: 1 IU/50 μg, 1 IU/60 μg, 1 IU/70 μg, 1 IU/80 μg, 1IU/90 μg, 1 IU/100 μg, 1 IU/125 μg, 1 IU/200 μg, 1 IU/250 μg, 1 IU/300μg, 1 IU/350 μg, 1 IU/400 μg, 1 IU/450 μg, 1 IU/500 μg, 1 IU/550 μg, 1IU/600 μg, 1 IU/700 μg, 1 IU/800 μg, 1 IU/1000 μg, 1 IU/1500 μg, 1IU/2000 μg, 1 IU/2500 μg, 1 IU/3000 μg, 1 IU/4000 μg, 1 IU/5000 μg, 1IU6000 μg, 1 IU/7000 μg, 1 IU/8000 μg, 1 IU/9000 μg, 1 IU/10000 μg, anda range between any two of the above ratios. In particular, the ratio ofsaid virus to said PIC is 1 IU/500 μg.

In some embodiments, the amount of PIC in the composition is from 250 μgto 5000 μg per unit dose; for example, the amount of the PIC is selectedfrom the group consisting of 250 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg,3000 μg, 4000 μg, 5000 μg per unit dose and a range between any two ofthe above amounts.

In some particular embodiments, the amount of PIC in the composition isfrom 500 μg to 4000 μg per unit dose, or from 1000 μg to 3000 μg perunit dose, or from 1000 μg to 2500 μg per unit dose.

When the composition of the present disclosure is applied to adults, theamount of PIC in the composition is selected from the group consistingof 500 μg, 1000 μg, 1500 μg, 2000 μg per unit dose and a range betweenany two of the above amounts. When the composition of the presentdisclosure is applied to minors (e.g., children), the amount of PIC inthe composition is selected from the group consisting of 250 μg, 500 μg,1000 μg, 1250 μg per unit dose and a range between any two of the aboveamounts.

In some embodiments, the unit dose in the present disclosure is preparedinto a volume which is selected from the group consisting of 0.1 ml,0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml,5.0 ml, 10.0 ml, 20.0 ml, 30.0 ml, 40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml,80.0 ml, 90.0 ml, 100.0 ml, 150.0 ml, 200.0 ml, 250.0 ml, and a rangebetween any two of the above volumes. It will be understood by theperson skilled in the art that an administration volume that is toolarge or too small leads to inconvenience in clinical practice.Therefore, when the compositions of the present disclosure areadministered to a human subject, the unit dose is preferably in therange of 0.5 ml to 1.0 ml for injection, preferably in the range of 0.15ml to 0.2 ml for intranasal administration, preferably in the range of30.0 ml to 100.0 ml for intravenous injection. It is understood thatalthough the unit dose is expressed in volume, this does not mean thatthe composition of the present disclosure can only be in a liquid dosageform. When the composition of the present disclosure is prepared in asolid dosage form (dry or lyophilized powder), the volume per unit doserefers to the solution volume after reconstitution of the dry orlyophilized powder.

In some embodiments, the amount of the virus in the composition is from0.1 IU to 100.0 IU per unit dose or from 0.2 to 100.0 IU per unit dose.In particular, the amount of the virus in the composition is selectedfrom the group consisting of 0.2 IU, 0.5 IU, 1.0 IU, 1.5 IU, 2.0 IU, 2.5IU, 3.0 IU, 3.5 IU, 4.0 IU, 5.0 IU, 6.0 IU, 7.0 IU, 8.0 IU, 9.0 IU, 10.0IU, 15.0 IU, 20.0 IU, 30.0 IU, 40.0 IU, 50.0 IU, 60.0 IU, 70.0 IU, 80.0IU, 90.0 IU, 100.0 IU per unit dose, and a range between any two of theabove amounts. In some particular embodiments, the amount of the virusin the composition is from 0.5 IU to 3.0 IU per unit dose; preferablyfrom 1.0 IU to 2.5 IU per unit dose. In some particular embodiments,when applied to adults, the amount of the virus in the composition isfrom 0.5 IU to 10.0 IU per unit dose. In some other particularembodiments, when applied to minors, the amount of the virus in thecomposition is from 0.5 IU to 5.0 IU per unit dose.

In some embodiments, the concentration of the virus in the compositionis from 0.05 IU/ml to 40.0 IU/ml; preferably selected from the groupconsisting of 0.05 IU/ml, 0.1 IU/ml, 0.15 IU/ml, 0.2 IU/ml, 0.5 IU/ml,1.0 IU/ml, 2.0 IU/ml, 3.0 IU/ml, 4.0 IU/ml, 5.0 IU/ml, 10 IU/ml, 15IU/ml, 20 IU/ml, 25 IU/ml, 30 IU/ml, 35 IU/ml, 40 IU/ml.

In some embodiments, the composition of present disclosure furthercomprises at least one auxiliary material as excipient and stabilizer.The auxiliary material is selected from the group consisting of gelatin,sucrose, sugar, lactose, maltose, trehalose, glucose, low molecularweight dextran, sorbitol, polysorbate 20, mannitol polyethylene glycol,human serum albumin, recombinant albumin, sodium octoate, urea, aluminumhydroxide, phenol red, magnesium chloride, potassium chloride, sodiumchloride, sodium thiosulfate, potassium dihydrogen phosphate, ascorbicacid, trichloromethane, phenol, and thimerosal and any combinationthereof.

In some embodiments, the composition of present disclosure furthercomprises at least one physiologically acceptable buffer which isselected from the group consisting of acetate,tris(hydroxymethyl)aminomethane (tris), bicarbonate, carbonate,phosphate buffer and any combination thereof. The pH of the buffer orcomposition is selected from the group consisting of 6.50, 6.60, 6.70,6.80, 6.90, 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45,7.50, 7.55, 7.60, 7.65, 7.70, 7.75, 7.80, 7.85, 7.90, 7.95, 8.00, and arange between any two of the above pH values. In one embodiment, the pHof the composition is between pH 6.5 to pH 8.0. In some particularembodiments, the buffer is PBS, and the pH of the buffer or compositionis from 7.3 to 7.5.

The composition of the present disclosure may be prepared in a soliddosage form or a liquid dosage form. The composition of presentdisclosure may be prepared in the form selected from the groupconsisting of a dry powder, a liquid solution or a liquid dosage form(e.g., injectable solution, aqueous or physiological saline solution,suspension, ointment, droplet, emulsion, gel, syrup or serofluid),tablet, coated tablet, microcapsule, suppository, granule, sugar-coatedtablet, capsule. The method of preparation is generally described inVaccine 4th Edition (Stanley A Plotkin et al., W. B. Saunders Company2003). Preferably, the composition of present disclosure is prepared inthe form of an injectable solution.

In another aspect, the present disclosure provides a composition for usein treatment of cancer, which is in the form of a dry powder orlyophilized powder.

In another aspect, the present disclosure provides the use of thecombination of PIC, at least one antibiotic (or at least one polyaminecompound) and at least one positive ion in the manufacture of amedicament for treatment of cancer.

In another aspect, the present disclosure provides the use of thecombination comprising a PIC adjuvant disclosed in CN103405762A in themanufacture of a medicament for treatment of cancer.

In another aspect, the present disclosure provides the use of thecombination of PIC, at least one antibiotic (or, at least one polyaminecompound), at least one positive ion and a virus in the manufacture of amedicament for treatment of cancer.

In another aspect, the present disclosure provides the use of thecomposition for treatment of cancer.

More particularly, the present disclosure provides the use of thecomposition in the manufacture of a medicament for treatment of cancer.

In some particular embodiments, the cancer is selected from the groupconsisting of:

oropharyngeal cancer, nasopharyngeal carcinoma, esophageal cancer,gastric cancer, colon cancer, liver cancer, cholangiocarcinoma,gallbladder cancer, pancreatic cancer, lung cancer, tracheal cancer,thymoma, bone cancer, joint cancer, melanoma, mesothelial cancer, breastcancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer,myleloma, blood cancer;

lip malignant neoplasm, tongue root malignant neoplasm, gingivalmalignant neoplasm, mouth malignant neoplasm, palate malignant neoplasm,parotid malignant neoplasm, tonsil malignant neoplasm, oropharyngealmalignant neoplasm, nasopharyngeal malignant neoplasm, piriform sinusmalignant neoplasm, hypopharynx malignant neoplasm;

esophagus malignant neoplasm, gastric malignant neoplasm, smallintestine malignant neoplasm, colon malignant neoplasm, rectosigmoidjunction malignant neoplasm, rectal malignant neoplasm, anal and analcanal malignant neoplasm, liver and intrahepatic bile duct malignantneoplasm, gallbladder malignant neoplasm, pancreatic malignant neoplasm;

nasal and middle ear malignant neoplasm, nasal sinus malignant neoplasm,laryngeal malignant neoplasm, tracheal malignant neoplasm, bronchial andpulmonary malignant neoplasm, thymus malignant neoplasm, heart,mediastinal and pleural malignant neoplasm;bone and articular cartilage malignant neoplasm;skin malignant melanoma;mesothelial and soft tissue malignant neoplasm;breast malignant neoplasm;genital malignant neoplasm, vaginal malignant neoplasm, cervicalmalignant neoplasm, uterine malignant neoplasm, ovarian malignantneoplasm, placental malignant neoplasm;penile malignant neoplasm, prostate malignant neoplasm, testicularmalignant neoplasm;urinary tract malignant neoplasm;eye and appendage malignant neoplasm;meningeal malignant neoplasm, brain malignant neoplasm;spinal cord, cranial nerve and central nervous system malignantneoplasm;endocrine gland malignant neoplasm;

Hodgkin's disease, follicular nodular non-Hodgkin's lymphoma, diffusenon-Hodgkin's lymphoma, peripheral and cutaneous T-cell lymphoma,multiple myeloma, malignant plasma cell tumor, lymphoid leukemia,myeloid leukemia, monocytic leukemia.

In some particular preferable embodiments, the composition of presentdisclosure is used in treatment of cancer which is selected from thegroup consisting of lung cancer, breast cancer, thyroid cancer, kidneycancer, gastric adenocarcinoma, liver cancer, melanoma, tongue cancer,rectal cancer, endometrial cancer, and ovarian cancer.

In a further preferable embodiment, the composition of presentdisclosure is used in treatment of metastatic tumor.

In further embodiments, the present disclosure provides the use of thecomposition in combination with an anti-tumor treatment regimen in themanufacture of a medicament for treatment of cancer;

preferably, said anti-tumor treatment regimen is selected from the groupconsisting of chemotherapy, radiotherapy, targeted therapy, andimmunotherapy, wherein therapeutical agent used in said chemotherapy isselected from the group consisting of alkylating agent, anti-metabolicantineoplastic agent, anti-tumor antibiotic, anti-tumor botanical,platinum compound antineoplastic agent, hormonal balance antineoplasticagent, and miscellaneous antineoplastic agent, wherein therapeuticalagent used in said targeted therapy is selected from the groupconsisting of rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine,cetuximab, nilotinib, and sorafenib, wherein therapeutical agent used insaid immunotherapy is selected from the group consisting of PD-1inhibitor, PD-L1 inhibitor and CTLA4 inhibitor; more preferably, saidalkylating agent is selected from the group consisting ofcyclophosphamide, ifosfamide and thiotepa, said anti-metabolicantineoplastic agent is selected from the group consisting ofmethotrexate, mercaptopurine, fluorouracil and cytarabine, saidanti-tumor antibiotic is selected from the group consisting ofbleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin andmitoxantrone, said anti-tumor botanical is selected from the groupconsisting of vincristine, etoposide, teniposide, paclitaxel anddocetaxel, said platinum compound antineoplastic agent is selected fromthe group consisting of cisplatin, carboplatin and oxaliplatin, saidhormone balance antineoplastic agent is selected from the groupconsisting of leuprolide, tamoxifen, flutamide and formestane, saidmiscellaneous antineoplastic agent is arsenic trioxide.

Alternatively, the present invention includes the use of a compositiondisclosed herein in the manufacture of a medicament for treatment ofcancer; wherein the medicament is for use in combination with ananti-tumour treatment regimen. Preferably, said anti-tumor treatmentregimen is selected from the group consisting of chemotherapy,radiotherapy, targeted therapy, and immunotherapy, wherein therapeuticalagent used in said chemotherapy is selected from the group consisting ofalkylating agent, anti-metabolic antineoplastic agent, anti-tumorantibiotic, anti-tumor botanical, platinum compound antineoplasticagent, hormonal balance antineoplastic agent, and miscellaneousantineoplastic agent, wherein therapeutical agent used in said targetedtherapy is selected from the group consisting of rituximab, bevacizumab,trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib,wherein therapeutical agent used in said immunotherapy is selected fromthe group consisting of PD-1 inhibitor, PD-L1 inhibitor and CTLA4inhibitor; more preferably, said alkylating agent is selected from thegroup consisting of cyclophosphamide, ifosfamide and thiotepa, saidanti-metabolic antineoplastic agent is selected from the groupconsisting of methotrexate, mercaptopurine, fluorouracil and cytarabine,said anti-tumor antibiotic is selected from the group consisting ofbleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin andmitoxantrone, said anti-tumor botanical is selected from the groupconsisting of vincristine, etoposide, teniposide, paclitaxel anddocetaxel, said platinum compound antineoplastic agent is selected fromthe group consisting of cisplatin, carboplatin and oxaliplatin, saidhormone balance antineoplastic agent is selected from the groupconsisting of leuprolide, tamoxifen, flutamide and formestane, saidmiscellaneous antineoplastic agent is arsenic trioxide.

The present invention also includes use of a composition disclosedherein the manufacture of a medicament for treatment of cancer and useof at least one further anti-tumour agent in the manufacture of amedicament for treatment of cancer. The present invention also includesuse of a composition disclosed herein and at least one furtheranti-tumour agent in the manufacture of a medicament for treatment ofcancer.

Preferably, the anti-tumour compound is selected from the groupconsisting of a chemotherapeutic agent, a targeted therapeutic agent andan immunotherapeutic agent.

Preferably, the chemotherapeutic agent is selected from the groupconsisting of alkylating agent, anti-metabolic antineoplastic agent,anti-tumor antibiotic, anti-tumor botanical, platinum compoundantineoplastic agent, hormonal balance antineoplastic agent, andmiscellaneous antineoplastic agent.

Preferably, said targeted therapeutical agent is selected from the groupconsisting of rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine,cetuximab, nilotinib, and sorafenib.

Preferably, said immunotherapeutical agent is selected from the groupconsisting of PD-1 inhibitor, PD-L1 inhibitor and CTLA4 inhibitor;

Preferably, said alkylating agent is selected from the group consistingof cyclophosphamide, ifosfamide and thiotepa.

Preferably, said anti-metabolic antineoplastic agent is selected fromthe group consisting of methotrexate, mercaptopurine, fluorouracil andcytarabine.

Preferably, said anti-tumor antibiotic is selected from the groupconsisting of bleomycin, daunorubicin, actinomycin D, mitomycin,doxorubicin and mitoxantrone.

Preferably, said anti-tumor botanical is selected from the groupconsisting of vincristine, etoposide, teniposide, paclitaxel anddocetaxel,

Preferably, said platinum compound antineoplastic agent is selected fromthe group consisting of cisplatin, carboplatin and oxaliplatin.

Preferably, said hormone balance antineoplastic agent is selected fromthe group consisting of leuprolide, tamoxifen, flutamide and formestane

Preferably, said miscellaneous antineoplastic agent is arsenic trioxide.

In the context of present disclosure, when the composition of presentdisclosure comprises a virus, the cancer to be treated is not caused bythe virus in the composition. In some particular embodiments, the cancerto be treated is not caused by rabies virus.

In another aspect, the present disclosure provides a method fortreatment of cancer, comprising a step of administering atherapeutically effective amount of the composition to a subject, forexample a human subject.

In some embodiments, the administration of the composition is systemicor localized. In some embodiments, the composition is administered viaparenteral injection (e.g., intramuscular, intraperitoneal, intravenous,subcutaneous, intradermal, intratumoral, peritumoral). In some otherembodiments, the composition is intradermally administered via a routeother than injection (e.g., a route that does not destroy the epithelialcell barrier by mechanical means). In some other embodiments, thecomposition is administered via a rectal, vaginal, nasal (for exampleintranasal), oral, buccal, sublingual, respiratory, ocular (for exampleintraocular), or transdermal route.

In particular, examples of the administration route includeintramuscular, intraperitoneal, intravenous, subcutaneous, transdermal,intradermal, intranasal, intraocular, oral, sublingual, intratumoral,and peritumoral.

In some embodiments, the composition of present disclosure isadministered to a human subject based on the frequency selected from thegroup consisting of once per month, 2 times per month, 3 times permonth, 4 times per month, 5 times per month, 6 times per month, 7 timesper month, 8 times per month, once per week, 2 times per week, 3 timesper week, 4 times per week, 5 times per week, 6 times per week, onceevery three days, 2 times every three days, 3 times every three days,once every two days, 2 times every two days, once per day, 2 times perday.

In particular, the method comprises administering a therapeuticallyeffective amount of a composition according to the invention to a humansubject through intramuscular injection at a frequency of 2 times everythree days, or administering a therapeutically effective amount of thecomposition according to the invention to a human subject throughintramuscular injection at a frequency of once per week.

In another aspect, the present disclosure provides a pharmaceutical kitor a kit for use in implementating said treatment method comprising atleast one container, wherein each of said container independentlycomprises the composition of present disclosure. The composition and/oramount of the composition in the different containers may be the same ordifferent.

In some embodiments, the kit of the present disclosure comprises atleast one container containing 2.0 IU/ml of inactivated rabies virus and1000 μg/ml of PIC.

In some embodiments, the composition of present disclosure is formulatedin sterile liquid and is contained in sterile container (e.g., tube,bottle, ampoule, syringe). In other embodiments, the composition ofpresent disclosure is contained in a container in the form of dry orlyophilized powder which is reconstituted into liquid form before use.

In some embodiments, the kit of present disclosure further comprises theitem selected from the group consisting of needle, water for injection,instruction for use, and any combination thereof.

Definitions

As used herein, the term “comprising” or “including” is to beinterpreted as specifying the presence of the stated features, integers,steps or components as referred to, but does not preclude the presenceor addition of one or more features, integers, steps or components, orgroups thereof. However, in context with the present disclosure, theterm “comprising” or “including” also includes “consisting of”. Thevariations of the word “comprising”, such as “comprise” and “comprises”,and “including”, such as “include” and “includes”, have correspondinglyvaried meanings.

“Inactivated” refers to the removal of virus pathogenicity and/orreprlicative capacity, but maintaining the ability to induce aprotective immune response in human body. Virus inactivation strategiesare known in the industry. Any common method can be used to inactivatethe virus, and a suitable method may be selected for a specific virustype. Virus inactivation methods including but not limited to: using aphotoreactive compound, an oxidizing agent, radiation (e.g., UVradiation, γ-radiation), UV radiation in combination with riboflavin,solvent/detergent (S/D) treatment (such as using tri(nbutyl)phosphateand/or Tween 80), polyethylene glycol (PEG) treatment, pasteurization(heat treatment), acidic pH treatment, enzyme treatment (pepsin ortrypsin), methylene blue light treatment, with dimethyl methylene blueand visible light, psoralen derivative S-59 and UVA irradiationtreatment.

“Attenuated”, An attenuated virus is still viable but the virulence isreduced during the manufacturing process. Attenuated virus retains theability to replicate and stimulate the human body to induce immuneresponse.

The term “heterogeneous” as used herein in the context of thecomposition of present disclosure indicates that the PIC molecules inthe composition are not uniform with respect to molecular weight, size,or both.

The term “unit dose” as used herein refers to physically discrete unitssuitable as unitary dosages for human subjects. Each unit contains apredetermined amount of present composition sufficient to produce thedesired effect in combination with the pharmaceutically/physiologicallyacceptable diluent or carrier.

The term “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacological and/orphysiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure of a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in subject,particularly a human, and includes: (i) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (ii) inhibiting the disease, i.e.,arresting its development; or (iii) relieving the disease, i.e., causingregression of the disease.

In the present disclosure, when describing numerical range, theexpression “ . . . to . . . ”, “within the range”, “arrange between” andthe like include the endpoint values.

As used herein, the term virus refers to a whole virus or a fragment ofa virus and includes a virus vaccine as well as a viral antigen.

The following examples and drawings and descriptions of the drawings areonly for the purpose of specifically illustrating the present invention,and is not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . The structure of PIC, antibiotic (or polyamine compound) andpositive ion observed under electron microscopy.

FIG. 2A. Tumor volume of LL/2 tumor-bearing mice after treatment.▪Vehicle control group, •Cisplatin group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. **: p<0.01 vs. vehiclecontrol group.

FIG. 2B. Relative tumor proliferation rate (T/C) of LL/2 tumor-bearingmice after treatment. •Cisplatin group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group.

FIG. 3 . Tumor weight of LL/2 tumor-bearing mice after treatment. **:p<0.01 vs. vehicle control group. #: p<0.05 vs. YS-ON-001 high dosegroup.

FIG. 4 . Body weight of LL/2 tumor-bearing mice after treatment.▪Vehicle control group, •Cisplatin group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. **: p<0.01 vs. vehiclecontrol group.

FIG. 5 . Body weight change of LL/2 tumor-bearing mice after treatment.▪Vehicle control group, •Cisplatin group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. *: p<0.05 vs. vehiclecontrol group. **: p<0.01 vs. vehicle control group.

FIGS. 6A & 6B. Thymus weight and thymus index of LL/2 tumor-bearing miceafter treatment. **: p<0.01 vs. vehicle control group.

FIGS. 7A & 7B. Spleen weight and spleen index of LL/2 tumor-bearing miceafter treatment. *: p<0.05 vs. vehicle control group.

FIG. 8 . Tumor volume of 4T1 tumor-bearing mice after treatment.▪Vehicle control group, •Docetaxel group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. **: p<0.01 vs. vehiclecontrol group. #: p<0.05 vs. YS-ON-001 high dose group. ##: p<0.01 vs.YS-ON-001 high dose group.

FIG. 9 . Relative tumor proliferation rate (T/C) of 4T1 tumor-bearingmice after treatment. •Docetaxel group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group.

FIG. 10 . Tumor weight of 4T1 tumor-bearing mice after treatment. Barrepresented SEM. **: p<0.01 vs. vehicle control group. #: p<0.05 vs.YS-ON-001 high dose group.

FIG. 11 . Body weight of 4T1 tumor-bearing mice after treatment.▪Vehicle control group, •Docetaxel group, ΔYS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. *: p<0.05 vs. vehiclecontrol group. **: p<0.01 vs. vehicle control group.

FIG. 12 . Body weight change of 4T1 tumor-bearing mice after treatment.▪Vehicle control group, •Docetaxel group, ⋄YS-ON-001 low dose group,⋄YS-ON-001 high dose group. Bar represented SEM. *: p<0.05 vs. vehiclecontrol group. **: p<0.01 vs. vehicle control group.

FIGS. 13A & 13B. Thymus weight and thymus index of 4T1 tumor-bearingmice after treatment. *: p<0.05 vs. vehicle control group. **: p<0.01vs. vehicle control group.

FIGS. 14A & 14B. Spleen weight and spleen index of 4T1 tumor-bearingmice after treatment. *: p<0.05 vs. vehicle control group. **: p<0.01vs. vehicle control group.

FIG. 15A. Tumor volume of H22 tumor-bearing mice during treatment. Barrepresented SEM. *: p<0.05 vs. vehicle control group. **: p<0.01 vs.vehicle control group. #: p<0.05 vs. Sorafenib+YS-ON-001 group. ##:p<0.01 vs. Sorafenib+YS-ON-001 group.

FIG. 15B. Relative tumor proliferation rate (T/C) of H22 tumor-bearingmice during treatment.

FIG. 16A. Tumor volume of LL/2 tumor-bearing mice after treatment. Barrepresented SEM. **: p<0.01 vs. vehicle control group.

FIG. 16B. Relative tumor proliferation rate (T/C) of LL/2 tumor-bearingmice after treatment.

FIG. 17 . Tumor weight of LL/2 tumor-bearing mice after treatment. Barrepresented SEM. **: p<0.01 vs. vehicle control group.

FIGS. 18A & 18B. Body weight of B16F10 tumor-bearing mice posttreatment. Bar represented SEM. *: p<0.05 vs. vehicle control group. **:p<0.01 vs. vehicle control group.

FIG. 19 . Number of lung metastases of B16F10 tumor-bearing mice posttreatment. Bar represented SEM. **: p<0.01 vs. vehicle control group.

FIG. 20A-20D. Thymus and spleen weight of B16F10 tumor-bearing mice posttreatment. Bar represented SEM. *: p<0.05 vs. vehicle control group. **:p<0.01 vs. vehicle control group.

FIG. 21 . Tumor volume of S180 tumor-bearing mice during treatment. Barrepresented SEM. **: p<0.01 vs. vehicle control group.

FIG. 22 . Tumor weight of S180 tumor-bearing mice after treatment. Barrepresented SEM. **: p<0.01 vs. vehicle control group.

FIG. 23 . Survival rate of S180 tumor-bearing mice during treatment.•Saline, ▪CTX, ♦YS-ON-001.

FIGS. 24A & 24B. Chest CT. FIG. 24A shows the CT scan beforeadministration of YS-ON-001; FIG. 24B shows the CT scan after twocourses of YS-ON-001 treatment.

EXAMPLES

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Unless otherwise stated, the animal studies described in this disclosurewere conducted in accordance with Regulations on the Administration ofLaboratory Animals, Guidance on the Care of Laboratory Animals, andChina National Standard GB/14925.

Example 1. The Manufacture Method of the Composition of PresentDisclosure

1. Virus:

Inactivated rabies virus.

2. The composition of present disclosure is prepared according to thefollowing components:

The composition comprises inactivated rabies virus, PIC, Kanamycin andcalcium chloride. The composition further comprises auxiliary materialsincluding maltose, dextran and human serum albumin.

Wherein, inactivated rabies virus: PIC=2.0 IU: 1000 μg.

Under sterile condition, the above composition is formulated in aphysiologically acceptable buffer. The concentration and volume of thecomposition to be formulated are allowed to be adjusted according to thefactors including subject to be administered (including but not limitedto age, sex, body weight, health condition), cancer condition (includingbut not limited to cancer type, severity), administration route, andadministration frequency. For the same therapeutically effective amount,when the concentration of the composition is high, the administrationvolume is small; when the concentration of the composition is low, theadministration volume is large.

When the composition is applied to mouse, the composition YS-ON-001 isprepared in the concentration of 2.0 IU/mL inactivated rabies virus,1000 μg/mL PIC, 800 IU/mL kanamycin and 0.16 μmol/mL calcium ion.

PIC is unstable in human body, and can be quickly broken down bynuclease. Antibiotic (or polyamine compound) and positive ion can form athree-dimensional structure with PIC, thereby increasing PIC stability.Electron microscope shows the final structure in FIG. 1 .

Example 2. Therapeutic Effects of the Composition (YS-ON-001) in LungCancer Animal Model

I. Construction of tumor model

1. Animals

1.1 Female C57BL/6 mice (6 weeks old, body weight 17.5±0.1 g) wereobtained from Shanghai SIPPR-BK Laboratory Animal Co. Ltd.

1.2 Animal Husbandry

Animals were fed according to the SPF level animal feeding conditions,and were provided with food (Keaoxieli certified Rodent Diet). Cage,bedding, feed and drinking water were autoclaved. Cages were placed inthe laminar flow rack with the cleanliness of 100, 3-5 animals per cage,and cages were replaced twice a week. The temperature in animal room is21 to 25° C., with the relative humidity between 40% and 70%.

2. Tumor Cell Line

The murine Lewis lung cancer cell line LL/2 (ATCC® CRL-1642™) wasobtained from American Type Culture Collection (ATCC). Cells weremaintained in DMEM with 10% fetal bovine serum in a atmosphere of 5% CO₂at 37° C. Passaging ratio was between 1:5 and 1:8, at the frequency of 3to 4 times per week.

3. Construction of LL/2 Tumor Transplanting Model

LL/2 murine Lewis lung tumor model was established in female C57BL/6mice by subcutaneous inoculation with 1×10⁶ cells per animal. The rateof tumor formation was 100%. Three days post cell inoculation, the cellinoculated mice were randomly sorted into treatment groups by weight.Animals in each group were administered according to Table 1.

II. Test Method

1. Test Groups

There were 4 groups: Vehicle group, Cisplatin group, YS-ON-001 low dosegroup, and YS-ON-001 high dose group.

TABLE 1 Experimental Design for LL/2 Route of Group Treatment NAdministration Dosing Level Schedule 1 Vehicle 10 i.m 0.2 mL/mouse q3d,total (negative 6 dosing control) 2 Cisplatin 10 i.v   5 mg/kg QW, total(positive control) 3 dosing 3 The composition 10 i.m 0.1 mL/mouse q3d,total YS-ON-001 6 dosing 4 The composition 10 i.m 0.2 mL/mouse q3d,total YS-ON-001 6 dosing Group 1: In vehicle group, DPBS was injectedinto both hind legs at 0.1 mL/site. Group 3: YS-ON-001 low dose group,0.1 mL of YS-ON-001 was injected into right hind leg and 0.1 mL of DPBSwas injected into left hind leg in 0,1 mL/mouse Group 4: YS-ON-001 highdose group, YS-ON-001 was injected into both hind legs at 0.1 mL/site,in total 0.2 mL.

2. The studies were terminated when the average tumor volume in vehiclegroup reached 2500 mm³

3. At the end of study, the tumor mass was removed and photographed. Thetumor weight was recorded. The thymus and spleen was removed andweighed. And then, the organ index was calculated.

III. Observational Indices

1. Tumor Volume

The anti-tumor effect of the test article was observed by measuringtumor diameter. Tumor volumes were measured 3 times weekly and therelative tumor proliferation rate T/C (%) was calculated.

1.1 Tumor volume (TV) was calculated as follow:

TV=½×a×b ²

where a and b denote long and wide, respectively.

1.2 Relative tumor proliferation rate T/C (%)=TV_(t)/TV_(c)×100%

Where TV_(t) is the mean tumor volume of the treatment group and TV_(c)is the mean tumor volume of the vehicle control group.

2. Tumor Weight

At the end of study, tumor mass was removed and weighed. Record thetumor weight and calculate the tumor inhibition rate according to thefollowing formula:

Tumor inhibition rate=(TW_(C)−TW_(T))/TW_(C)×100%

Where TW_(t) is the mean tumor weight of the treatment group and TV_(c)is the mean tumor weight of the vehicle control group.

3 Body Weight

Body weight of each mouse was weighed three times a week. Body weightchange (BWC) was calculated as:

BWC=(BW_(n)−BW₀)/BW₀×100%

Where BWn is the BW at Day n and BW₀ is the BW at the day when thetreatment was initiated.

4 Thymus and Spleen Weight

At the end of study, thymus and spleen was removed and weighed. Organindex was calculated as: organ index=organ weight/body weight×100%.

IV. Data Presentation and Analysis

Data were reported as Mean±SEM. Comparison between groups was analysedusing ANOVA.

V. Results

1. Tumor Volume

A graphical presentation of the effect of YS-ON-001 on LL/2 murine Lewislung tumor volume is shown in FIG. 2A.

A graphical presentation of the effect of YS-ON-001 on LL/2 murine Lewislung tumor proliferation rate is shown in FIG. 2B.

Take the day of the tumor as Day 0. On Day 3, animals were randomized bybody weight and administered according to group assignment.

Tumors in the vehicle control group grew progressively in all ten mice.At the end of study, the mean tumor volume reached 2707±257 mm³.

Treatment with Cisplatin significantly inhibited the tumor growthstarting from Day 10 till end of the study, resulting in a relativetumor proliferation rate of 53.04%, with a significant differencecompared to the vehicle control group. (p<0.01).

YS-ON-001 treatment significantly inhibit tumor growth starting from Day10 till end of the study compared to vehicle control group (p<0.01). Thetumor volume in high dose group is lower than that in low dose group,however the difference is not significant (p>0.05). The relative tumorproliferation rate at the end of the study is 43.36% and 34.33%,respectively.

2. Tumor Weight

A graphical presentation of the effect of YS-ON-001 on LL/2 murine Lewislung tumor weight is shown in FIG. 3 .

At the end of study, the tumor mass was removed and weighed. Similarwith tumor volume, Treatment with Cisplatin or YS-ON-001 significantlylower the tumor weight compared to the vehicle control group (p<0.01).The tumor weight in YS-ON-001 high dose group is significantly lowerthan that in Cisplatin group and YS-ON-001 low dose group (p<0.05). Theinhibition rate is 29.46%, 29.49% and 53.87%, for Cisplatin group,YS-ON-001 low dose and high dose group, respectively.

3. Body Weight

A graphical presentation of the effect of YS-ON-001 on body weight andbody weight changes of LL/2 murine Lewis lung tumor is shown in FIGS. 4& 5 .

The average body weight (BW) was significantly affected by the treatmentof Cisplatin compared to the vehicle group on Day 5 and from Day 10 toend of study (p<0.01). The same result was observed on the body weightchange.

The mouse body weight was not obviously effected by the treatment of twoYS-ON-001 groups compared to vehicle group (p>0.05). The body weightchange in YS-ON-001 high dose group was significantly lower on Day 7compared to vehicle control group (p<0.05), while there was nosignificant change at other time points.

4. Thymus and Spleen Weight

A graphical presentation of the thymus and spleen weight as well asorgan index of LL/2 murine Lewis lung tumor is shown in FIGS. 6 & 7 .

At the end of study, except tumor mass, the thymus and spleen wasremoved and weighed. Compared to the vehicle group, Cisplatin treatmentsignificantly reduced thymus weight and spleen weight. The thymus weightand thymus index is significantly reduced compared to that in vehiclecontrol group (p<0.01). The spleen weight is significantly reducedcompared to that in vehicle control group (p<0.05), however the spleenindex shows a trend of reducing which is not significant (p>0.05). Thethymus weight, spleen weight and their organ index were not affected bythe treatment of YS-ON-001 compared to the vehicle group.

VI. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the cisplatin group showed a significant inhibitory effect ontumor growth as a positive control.

YS-ON-001 showed anti-tumor activity in a dose-dependent manner whenused as a single agent at 0.1 mL/mouse and 0.2 mL/mouse.

Moreover, the animals were well tolerated and showed no significanteffect on body weight, spleen weight and thymus weight. The side effectswere significantly lower than those in Cisplatin group. The antitumoreffect in YS-ON-001 high dose group was better than that in Cisplatingroup.

The results showed that YS-ON-001 showed inhibitory effect in adose-dependent manner on tumor growth at the tested dose level in LL/2Lewis lung cancer animal model. Meanwhile the tumor-bearing mice showedno obvious side effects.

Example 3. Therapeutic Effects of the Composition (YS-ON-001) in BreastCancer Animal Model

I. Construction of Tumor Model

1. Animals

1.1 Female Balb/c mice (6 weeks old, body weight 17.2±0.1 g) wereobtained from Shanghai SIPPR-BK Laboratory Animal Co. Ltd.

2.2. Animal Husbandry

Animals were fed according to the SPF level animal feeding conditions,and were provided with food (Keaoxieli certified Rodent Diet). Cage,bedding, feed and drinking water were autoclaved. Cages were placed inthe laminar flow rack with the cleanliness of 100, 3-5 animals per cage,and cages were replaced twice a week. The temperature in animal room is21 to 25° C., with the relative humidity between 40% and 70%.

2. Tumor Cell Line

The murine breast cancer cancer cell line 4T1 was obtained from AmericanType Culture Collection (ATCC), and maintained by HD Biosciences. Cellswere maintained in DMEM with 10% fetal bovine serum in a atmosphere of5% CO₂ at 37° C. Passaging ratio was between 1:5 and 1:8, at thefrequency of 3 to 4 times per week.

3. Construction of 4T1 Tumor Transplanting Model

4T1 murine breast tumor model was established in female Balb/c mice bysubcutaneous inoculation with 1×10⁶ cells per animal. The rate of tumorformation was 100%. Three days post cell inoculation, the cellinoculated mice were randomly sorted into treatment groups by weight.Animals in each group were administered according to Table 2.

TABLE 2 Experimental Design for 4T1 Route of Group Treatment NAdministration Dosing Level Schedule 1 Vehicle 10 i.m 0.2 mL/mouse q3d,total (negative 6 dosing control) 2 Docetaxel 10 i.v  10 mg/kg QW, total(positive control) 3 dosing 3 The composition 10 i.m 0.1 mL/mouse q3d,total YS-ON-001 6 dosing 4 The composition 10 i.m 0.2 mL/mouse q3d,total YS-ON-001 6 dosing Group 1: In vehicle group, DPBS was injectedinto both hind legs at 0.1 mL/site. Group 3: YS-ON-001 low dose group,0.1 mL of YS-ON-001 was injected into right hind leg and 0.1 mL of DPBSwas injected into left hind leg in 0,1 mL/mouse Group 4: YS-ON-001 highdose group, YS-ON-001 was injected into both hind legs at 0.1 mL/site,in total 0.2 mL.

II. Test Method

1. Test groups

There were 4 groups: Vehicle group, Docetaxel group, YS-ON-001 low dosegroup, and YS-ON-001 high dose group.

2. The studies were terminated when the average tumor volume in vehiclegroup reached 2500 mm³

3. At the end of study, the tumor mass was removed and photographed. Thetumor weight was recorded. The thymus and spleen was removed andweighed. And then, the organ index was calculated.

III. Observational Indices: Same as in Example 2 Section III.

IV. Data Presentation and Analysis

Data were reported as Mean±SEM. Comparison between groups was analysedusing ANOVA.

V. Results

1. Tumor Volume

A graphical presentation of the effect of YS-ON-001 on 4T1 murine breasttumor volume is shown in FIG. 8 .

A graphical presentation of the effect of YS-ON-001 on 4T1 murine breasttumor proliferation rate is shown in FIG. 9 .

Take the day of the tumor as Day 0. On Day 3, animals were randomized bybody weight and administered according to group assignment.

Tumors in the vehicle control group grew progressively in all ten mice.At the end of study, the mean tumor volume reached 2576±108 mm³.

Treatment with Docetaxel significantly inhibited the tumor growthstarting from Day 8 till end of the study, resulting in a relative tumorproliferation rate of 50.12%, with a significant difference compared tothe vehicle control group. (p<0.01).

YS-ON-001 treatment significantly inhibit tumor growth starting from Day8 till end of the study compared to vehicle control group (p<0.01).There is a significant difference between YS-ON-001 low dose group andhigh dose group from Day 11 till end of the study (p<0.05). The relativetumor proliferation rate at the end of the study is 63.43% and 45.87%,respectively.

2. Tumor Weight

A graphical presentation of the effect of YS-ON-001 on 4T1 murine breasttumor weight is shown in FIG. 10 .

At the end of study, the tumor mass was removed and weighed. Similarwith tumor volume, Treatment with Docetaxel significantly lower thetumor weight compared to the vehicle control group (p<0.01). YS-ON-001high dose group significant decreased tumor weight when compared to thevehicle control group (p<0.01). YS-ON-001 low dose group slightlylowered tumor weight when compared to the vehicle control group. Thetumor weight in YS-ON-001 high dose group is significantly lower thanthat in YS-ON-001 low dose group (p<0.05). The inhibition rate is35.55%, 18.21%, and 42.26%, for Docetaxel group, YS-ON-001 low dose andhigh dose group, respectively.

3. Body Weight

A graphical presentation of the effect of YS-ON-001 on body weight andbody weight changes of 4T1 murine breast tumor is shown in FIGS. 11 & 12.

The average body weight (BW) was significantly affected by the treatmentof Docetaxel compared to the vehicle group on Day 8 and from Day 13 toend of study (p<0.01). The average body weight change was significantlyreduced by the treatment of Docetaxel compared to the vehicle group fromDay 4 to end of study (p<0.05). The weight at the end of the study alsoshowed a downward trend compared to that in the same group at the timeof grouping.

The mouse body weight was not obviously effected in YS-ON-001 low dosegroup compared to vehicle group. The body weight in YS-ON-001 high dosegroup was significantly lower from Day 15 than that in vehicle group(p<0.05). The body weight change in two YS-ON-001 groups wassignificantly lower on Day 4 and from Day 15 to end of study compared tovehicle control group (p<0.05 for low dose group, p<0.01 for high dosegroup). However the body weight was still increased compared to that inthe same group at the time of grouping.

4. Thymus and Spleen Weight

A graphical presentation of the thymus and spleen weight as well asorgan index of 4T1 murine breast tumor is shown in FIGS. 13 & 14 .

At the end of study, except tumor mass, the thymus and spleen wasremoved and weighed.

Compared to the vehicle group, Docetaxel treatment significantly reducedthymus and spleen weight and thymus and spleen index (p<0.01). Thethymus weight, spleen weight and thymus organ index were not affected bythe treatment of YS-ON-001 compared to the vehicle group. In both doseof YS-ON-001 groups, the spleen organ index was increased compared tothe vehicle group significantly (p<0.01 for low dose group, p<0.05 forhigh dose group).

VI. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the Docetaxel group showed a significant inhibitory effect ontumor growth as a positive control.

YS-ON-001 showed anti-tumor activity in a dose-dependent manner whenused as a single agent at 0.1 mL/mouse and 0.2 mL/mouse. Moreover, theanimals were well tolerated and showed no significant effect on thymusweight. The antitumor effect of high dose group was comparable to thatof Docetaxel group, with significantly reduced side effects.

In the present example, the spleen index in two YS-ON-001 groupsincreased compared to that in vehicle control group. Further analysis isneeded to understand the cause. Preliminary judgment may be enhancedimmune function resulting weight increasing in immune organ.

The results showed that YS-ON-001 showed inhibitory effect in adose-dependent manner on tumor growth at the tested dose level in 4T1breast cancer animal model. Meanwhile the tumor-bearing mice showed noobvious side effects.

Example 4. Therapeutic Effects of the Composition (YS-ON-001) Alone orCombined with Sorafenib in Subcutaneous H22 Murine HepatocellularCarcinoma Model

I. Construction of Tumor Model

1. Animals and animal husbandry were the same as in Example 2.

2. Tumor Cell Line

The murine hepatocellular carcinoma cell line H22 was obtained fromChina Center for Type Culture Collection (CCTCC) and maintained by HDBiosciences. Cells were maintained in Balb/c mice as ascites. Thepassage interval is 7-8 days.

3. Construction of H22 Tumor Transplanting Model

The donor mice with H22 ascites were sacrificed and sterilized by 75%alcohol immersion. The ascites was collected and re-suspended inpre-cooled DPBS at appropriate density. H22 murine hepatocellularcarcinoma tumor model was established in female Balb/c mice bysubcutaneous inoculation with 5×10⁶ cells per animal. The rate of tumorformation was 100%. Three days post cell inoculation, the cellinoculated mice were randomly sorted into treatment groups by weight.Animals in each group were administered according to Table 3.

II. Test Method

1. Test groups

There were 4 groups: Vehicle group, Sorafenib group, YS-ON-001 group,and Sorafenib+YS-ON-001 group.

TABLE 3 Experimental Group and Dosage Route of Group Treatment# NAdministration* Dosing Level Schedule 1 Vehicle* 10 i.m./s.c 0.2 mL/miceBID 2 Sorafenib 10 p.o.  60 mg/kg QD 3 The composition 10 i.m./s.c 0.2mL/mice BID YS-ON-001 Sorafenib + the  60 mg/kg + 4 composition 10 p.o +i.m./s.c 0.2 mL/mice BID + QD YS-ON-001 *Vehicle was DPBS. #Sorafenibwas administration via oral gavage, vehicle and YS-ON-001 was injected0.1 mL by i.m. and 0.1 mL by s.c..

2. The studies were terminated when the average tumor volume in vehiclegroup reached 2500 mm³

3. At the end of study, the tumor mass was removed and photographed. Thetumor weight was recorded.

III. Observational Indices: Same as in Example 2.

IV. Results

1. Tumor Volume

A graphical presentation of the effect of YS-ON-001on H22 murinehepatocellular carcinoma tumor volume is shown in FIG. 15A.

A graphical presentation of the effect of YS-ON-001on H22 murinehepatocellular carcinoma tumor proliferation rate is shown in FIG. 15B.

Take the day of the tumor as Day 0. On Day 3, animals were randomized bybody weight and administered according to group assignment.

Tumors in the vehicle-treated control group grew progressively in allten mice. When the vehicle group was terminated on Day 12, the meantumor volume reached 2120±182 mm³.

One mouse in the vehicle group and two mice in Sorafenib group weresacrificed at Day 10 and Day 19 when their tumor volume reached 2500mm³. The whole study was terminated on Day 21.

Treatment with Sorafenib significantly inhibited tumor growth from Day 3to end of the study compared to that in vehicle control group (p<0.01).The relative tumor proliferation rate on Day 12 is 29.90%.

Treatment with Sorafenib+YS-ON-001 significantly inhibited tumor growthfrom Day 1 to end of the study compared to that in vehicle control group(p<0.01). The relative tumor proliferation rate on Day 12 is 10.61%.

There are significant differences in tumor volume between Sorafenibgroup and Sorafenib+YS-ON-001 group on Day 1, Day 5 and from Day 10 toend of the study (p<0.05 or p<0.01).

There are significant differences in tumor volume between YS-ON-001group and Sorafenib+YS-ON-001 group on Day 5 and from Day 10 to end ofthe study (p<0.05 or p<0.01).

V. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the Sorafenib group showed a significant inhibitory effect ontumor growth as a positive control. These indicate that the results arereliable.

YS-ON-001 showed strong anti-tumor activity when used BID at 0.2mL/mouse. Sorafenib+YS-ON-001 showed a very significant inhibition oftumor growth and a significant synergistic effect. There were no animaldead due to drug toxicity in single drug group or combination druggroup.

The results showed that The YS-ON-001 and combination group showedstrong anti-tumor activity in H22 murine hepatocellular carcinoma tumormouse model at the tested dosing level. Combining YS-ON-001 andSorafenib induced strongly enhanced antitumor activity compared witheither agent alone.

Example 5. Therapeutic Effects of the Composition (YS-ON-001) and PIKAAlone in Subcutaneous LL/2 Murine Lewis Lung Cancer Model in FemaleC57BL/6 Mice

I. Construction of Tumor Model

1. Animals and animal husbandry were the same as in Example 2

2. The murine Lewis lung cancer cell line LL/2 was obtained, culturedand the LL/2 murine Lewis lung tumor model was established the same asin Example 2. Animals in each group were administered according to Table4.

II. Test Method

1. Test groups

There were 6 groups: Vehicle group, Cisplatin group, YS-ON-001 group,YS-ON-002 (no virus) group, Rabies vaccine group and Cisplatin+YS-ON-001group. The difference between YS-ON-001 and YS-ON-002 is that there isno inactivated rabies virus in YS-ON-002.

TABLE 4 Experimental Group and Dosage Route of Dosing Group Treatment NAdministration Level* Schedule 1 Vehicle 10 i.m. 0.2 mL/mouse Q2D, total7 times 2 Cisplatin 10 i.v. 5 mg/kg Q2D, total 2 times 3 The 10 i.m. 0.2mL/mouse Q2D, total composition 7 times YS-ON-001 4 YS-ON-002 10 i.m 0.2mL/mouse Q2D, total (PIKA alone) 7 times 5 Rabies 10 i.m. 0.2 mL/mouseQ2D, total Vaccine (Vero 7 times Cell) 6 Cisplatin + the 10 i.v. + i.m.0.2 mL/mouse QW (total 2 composition times) + Q2D YS-ON-001 (total 7times) *In vehicle group, DPBS was injected into both hind legs at 0.1mL/site. YS-ON-001 and YS-ON-002 were injected into both hind legs at0.1 mL/site.

2. The studies were terminated when the average tumor volume in vehiclegroup reached 2500 mm³

3. At the end of study, the tumor mass was removed and photographed. Thetumor weight was recorded.

III. Observational Indices

1. Tumor Volume

The measure and calculation of tumor volume, tumor weight were same asin Example 2.

2. Combination drug index

Q value was calculated according to Jin's formula, Q=0.85-1.15 for theadditional effect, Q>1.15 for the synergetic effect:

Q=Ea+b/(Ea+Eb−Ea×Eb)

Where Ea+b is the tumor inhibition rate in drug combination group, Eaand Eb are the tumor inhibition rates in single drug group.

IV. Results

1. Tumor Volume

A graphical presentation of the effect of YS-ON-001, YS-ON-002 and thecombination group on tumor volume of the mice bearing LL/2 murine Lewislung carcinoma is shown in FIG. 16A.

A graphical presentation of the effect of YS-ON-001, YS-ON-002 and thecombination group on tumor proliferation rate of the mice bearing LL/2murine Lewis lung carcinoma is shown in FIG. 16B.

Take the day of the tumor as Day 0. On Day 3, animals were randomized bybody weight and administered according to group assignment.

Tumors in the vehicle control group grew progressively in all ten mice.When the vehicle group was terminated, the mean tumor volume reached2535±148 mm³.

Treatment with Cisplatin significantly inhibited the tumor growthcompared with the vehicle control group from day 5 to the end of thestudy (p<0.01), resulting in a relative tumor proliferation rate of47.46%.

Treatment with YS-ON-001 and YS-ON-002 significantly inhibited tumorgrowth when compared to the vehicle control group from day 5 to the endof study (p<0.01). The relative tumor proliferation rate was 37.02% and40.56%, respectively.

Treatment with Rabies Vaccine had no obvious affect compared withvehicle control group (p>0.05). The relative tumor proliferation ratewas 97.87% at the end of the study.

The combination group significantly inhibited tumor growth when comparedwith the vehicle control group from day 5 to the end of the study(p<0.01). The relative tumor proliferation rate was 28.38% at the end ofthe study. The relative tumor proliferation rate was lower than that inCisplatin and YS-ON-001 alone groups. According to Jin's formula,YS-ON-001 and Cisplatin had additional effect, Q=0.87.

The tumor volume of YS-ON-001 group is lower than YS-ON-002 group,however the difference is statistically insignificant. The tumor volumeof YS-ON-001 group is lower than YS-ON-002 group from Day 5 to end ofthe study.

According to Jin's formula, rabies virus and other components in thecomposition have additional effect, Q=0.93.

2 Tumor Weight

A graphical presentation of the effect of YS-ON-001, YS-ON-002 and thecombination group on tumor weight of the mice bearing LL/2 murine Lewislung carcinoma is shown in FIG. 17 .

At the end of study, the tumor mass was removed and weighed. Similar tothe effect on tumor volume, Cisplatin, YS-ON-001, YS-ON-002 and thecombination group had significantly decreased tumor weight when comparedwith the vehicle control group (p<0.01), resulting in 42.38% 60.88,56.04% and 75.44% tumor inhibition rates, respectively.

According to Jin's formula, rabies virus and other components in thecomposition have additional effect, Q=0.97.

V. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the cisplatin group showed a significant inhibitory effect ontumor growth as a positive control. These indicate that the results arereliable.

YS-ON-001 and YS-ON-002 showed strong anti-tumor activity when used as asingle agent at 0.2 mL/mouse administrated i.m on LL/2 murine Lewis lungtumor model. The animals were well tolerated and had a slight effect onthe body weight, however the toxic side effects were significantly lowerthan the control drug Cisplatin. The anti-tumor effect was comparablewith the control drug Cisplatin.

The combination group's relative tumor proliferation rate was 28.38%,which was lower than the groups treated with Cisplatin and YS-ON-001alone. According to Jin's formula, YS-ON-001 and Cisplatin hadadditional effect.

There are no statistically significant variations in tumor inhibiteffect between YS-ON-001 and YS-ON-002, however rabies virus and othercomponents in the composition have additional effect according to Jin'sformula.

The results showed that YS-ON-001 and YS-ON-002 showed inhibitory effectat the tested dose level in LL/2 Lewis lung cancer animal model. Thecomposition YS-ON-001 and Cisplatin had additional effect

Example 6. Therapeutic Effects of the Composition (YS-ON-001) in B16F10Tumor Metastasis Model in Female C57BL/6 Mice

I. Construction of Tumor Model

1. Animals and animal husbandry were the same as in Example 2

2. Tumor Cell Line

The cell line B16F10 was obtained from American Type Culture Collection(ATCC) and maintained by HD Biosciences. Cells were maintained in DMEMwith 10% fetal bovine serum in an atmosphere of 5% CO₂ at 37° C.Passaging ratio was between 1:5 and 1:8, at the frequency of 3 to 4times per week.

on a 12-hour light/dark cycle.

3. Construction of LL/2 Tumor Transplanting Model

B16F10 metastasis tumor model was established in female C57BL/6 mice byintravenous inoculation with 5×10⁴/0.2 mL per animal. One day post cellinoculation, mice were randomly sorted into treatment groups by weight.Animals in each group were administered according to Table 5.

II. Test Method

1. Test groups

There were 3 groups: Vehicle group, Cisplatin group and YS-ON-001 group.

TABLE 5 Experimental Group and Dosage Route of Group Treatment NAdministration^(#) Dosing Level Schedule 1 Vehicle* 10 i.m./s.c. 0.2mL/mice/dose BID 2 Cisplatin 10 i.v. 5 mg/kg Q4D 3 The 10 i.m./s.c. 200μg/0.2 BID composition mL/mice/dose YS-ON-001 *Vehicle is DPBS^(#)Animals in vehicle group and YS-ON-001 group was dosed of 0.1 mL viai.m. (hind leg) and s.c. injection, respectively.

2. At the end of the study, the studies were terminated on day 15. Thelung was removed and photographed. The number of metastasis spot wascounted. The thymus and spleen were removed, weighed and calculated fororgan indices.

III. Observational Indices

1. Body Weight

Body weight of each mouse was weighed three times a week. Body weightchange (BWC) was calculated as:

BWC=(BW_(n)−BW₀)/BW₀×100%

Where BWn is the BW at Day n and BW₀ is the BW at the day when thetreatment was initiated.

2. Number of Lung Metastases

At the end of study, the animals were sacrificed and the lung wasremoved. And then, the metastasis number was counted.

3. Thymus and Spleen Weight

At the end of the study, the thymus and the spleen were removed andweighed.

IV. Data Presentation and Analysis

Data were reported as Mean±SEM. Comparison between groups was analysedusing ANOVA.

V. Results

1. Body Weight

A graphical presentation of the effect of YS-ON-001 on body weight andbody weight changes of the mice is shown in FIGS. 18A & 18B.

The average body weight (BW) was significantly lowered by the treatmentof Cisplatin compared with the vehicle group (p<0.01) from Day 5 to theend of the study (except day 7). A similar result was observed in thebody weight change.

In YS-ON-001 group, the body weight was transiently slightly loweredcompared to vehicle control group. However, on Day 5 and Day 15, thebody weight in YS-ON-001 group was significantly lowered compared tovehicle control group.

2 Number of Lung Metastases

A graphical presentation of the effect of YS-ON-001 on number of lungmetastasis of the mice is shown in FIG. 19 .

At the end of study, the animals were sacrificed and the lung wasremoved. And then, the metastasis number was counted.

Compared with vehicle group, the number of lung metastases significantlydecreased by treatment with Cisplatin and YS-ON-001 (p<0.01).

3 Thymus and Spleen Weight

A graphical presentation of the thymus and spleen weight as well as eachorgan index of the mice is shown in FIG. 20 .

At the end of the study, in addition to lung, the thymus and spleen wereremoved and weighed. The organ indices for each organ were calculated.

Compared with the vehicle group, treatment with Cisplatin significantlydecreased thymus weight (p<0.01), thymus index (p<0.01), while spleenweight and spleen index was not affected.

Compared with the vehicle group, the thymus weight and thymus index wassignificantly decreased by treatment with YS-ON-001 (p<0.01), while thespleen weight and spleen index was significantly increased (p<0.01).

VI. Discussion and Conclusion

In the present example, the lung metastases were observed in all animalsin vehicle control group, resulting in the average number of lungmetastases at 93.43±1.76. The average number of lung metastases wassignificantly reduced in Cisplatin group. These indicate that theresults are reliable.

The YS-ON-001 showed strong anti-metastasis activity with significantlyreduced average number of lung metastases in B16F10 tumor metastasismodel at the tested dosing level.

Example 7. Therapeutic Effects of the Composition (YS-ON-001) in S180Tumor Model in Female Balb/C Mice

I. Construction of Tumor Model

1. Animals and animal husbandry were the same as in Example 2

2. Tumor Cell Line

The cell line S180 was obtained from the Cell Bank of Chinese Academy ofMedical Sciences and maintained in IPE-CAS laboratory. Cells weremaintained in Balb/c mice as ascites. The passage interval is 5-6 days.

3. Construction of S180 Tumor Transplanting Model

Balb/c animals were fed for 5 to 7 days, and S180 tumor wasintraperitoneal inoculated into the F0 generation mice. The ascites weretaken out when the abdominal cavity to a certain extent, and theninoculated in F1 generation mice. The tests used F2-F3 generation mice.S180 tumor model was established in female Balb/c mice by subcutaneousinoculation with cell suspension (2×10⁶/0.2 mL/animal). Two days postcell inoculation, the cell inoculated mice were randomly sorted intotreatment groups. Animals in each group were administered according toTable 6.

II. Test Method

1. Test groups

There were 3 groups: Vehicle group, Cyclophosphamide (CTX) group, andYS-ON-001 group.

TABLE 6 Experimental Group and Dosage Route of Group Treatment NAdministration Dosing Level Schedule 1 Normal 10 i.m. 0.2 mL/mice/doseQ2D, in Saline total 10 times 2 Cyclophosphamide 10 i.p. 20 mg/kg Q2D,in (CTX) total 10 times 3 The 10 i.m 0.2 mL/mice/dose Q2D, incomposition total YS-ON-001 10 times

2. At the end of the study, the studies were terminated on day 21. Thetumor was removed and photographed. The weight of tumor was recorded.

III. Observational Indices

1. Tumor Volume

The anti-tumor effect of the test article was observed by measuringtumor diameter. Tumor volumes were measured 3 times weekly and therelative tumor proliferation rate T/C (%) was calculated.

1.1 Tumor volume (TV) was calculated as follow:

TV=½×a×b ²

where a and b denote long and wide, respectively.

2. At the end of study, tumor mass was removed and weighed.

IV. Data Presentation and Analysis

Data were reported as Mean±SEM. Comparison between groups was analysedusing ANOVA.

V. Results

1. Tumor Volume

A graphical presentation of the effect of YS-ON-001 group on tumorvolume of S180 tumor model in female Balb/C mice is shown in FIG. 21 .

Tumors in the saline-treated control group grew progressively in all tenmice. At day 21, the mean tumor volume reached 2000 mm³.

Treatment with CTX significantly inhibited the tumor growth comparedwith the saline control group from day 4 to the end of the study(p<0.01).

Treatment with YS-ON-001 significantly inhibited tumor growth whencompared to the saline control group from day 4 to the end of study(p<0.01). The tumor volume in YS-ON-001 group was similar to that in CTXgroup.

2 Tumor Weight

A graphical presentation of the effect of YS-ON-001 on tumor weight ofS180 tumor model in female Balb/C mice is shown in FIG. 22 .

At the end of study, the tumor mass was removed and weighed. Similar tothe effect on tumor volume, CTX and YS-ON-001 had significantlydecreased tumor weight when compared with the saline control group(p<0.01).

VI. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the CTX group showed a significant inhibitory effect on tumorgrowth as a positive control.

Treatment with YS-ON-001 significantly inhibited tumor growth withsignificantly reduced tumor volume and tumor weight.

The results showed YS-ON-001 showed strong anti-tumor activity in S180tumor model at the tested dosing level.

Example 8. Therapeutic Effects of the Composition (YS-ON-001) in EhrlichAscites Cancer (EAC) Tumor Model in Female Balb/C Mice

I. Construction of Tumor Model

1. Animals

1.1 Female Kunming mice (5-6 weeks old) were obtained and tested after5-6 days adaptive feeding.

1.2 Animal Husbandry

Animals were feed according to the SPF level animal feeding conditions,and were provided with food (Keaoxieli certified Rodent Diet). Cage,bedding, feed and drinking water were autoclaved. Cages were placed inthe laminar flow rack with the cleanliness of 100, 3-5 animals per cage,and cages were replaced twice a week. The temperature in animal room is21 to 25° C., with the relative humidity between 40% and 70%.

2. Tumor Cell Line

The Ehrlich Ascites Cancer (EAC) tumor cell line EAC was obtained fromthe Cell Bank of Chinese Academy of Medical Sciences and maintained inIPE-CAS laboratory. Cells were maintained in Kunming mice as ascites.The passage interval is 7-8 days.

3. Construction of Tumor Transplanting Model

Animals were fed for 5 to 7 days, and EAC tumor was intraperitonealinoculated into the F0 generation mice. The ascites were taken out whenthe abdominal cavity to a certain extent, and then inoculated in F1generation mice. The tests used F2-F3 generation mice. EAC tumor modelwas established in female Kunming mice by intraperitoneal inoculationwith cell suspension (1×10⁶/0.2 mL/animal). Two days post cellinoculation, the cell inoculated mice were randomly sorted intotreatment groups. Animals in each group were administered according toTable 7.

TABLE 7 Experimental Design for EAC Route of Group Treatment NAdministration Dosing Level Schedule 1 Vehicle 10 i.m. 0.2 mL/mouse Q2d,total 10 dosing 2 CTX 10 i.m. 20 mg/kg Q2d, total 10 dosing 3 The 10i.m. 0.2 mL/mouse Q2d, total composition 10 dosing YS-ON-001 Group 1: Invehicle group, PBS was injected into both hind legs at 0.1 mL/site.Group 3: YS-ON-001 group, YS-ON-001 was injected into both hind legs at0.1 mL/site, in total 0.2 mL

II. Test Method

1. Test groups There were 3 groups: Vehicle group, CTX group, andYS-ON-001 group.

2. At the end of the study, the studies were terminated on day 21. Thetumor was removed and photographed. The weight of tumor was recorded.

III. Observational Indices: Duration, Tumor Growth Delay

Tumor growth delay (TGD) was calculated as (TGD)=(T−C)/C×100%, where Tand C refer to the median duration (days) of individual mouse to die forthe treated and control groups, respectively.

IV. Data Presentation and Analysis

Comparison between groups was analysed using ANOVA.

V. Results

A graphical presentation of the survival rate of EAC tumor model infemale Kunming mice is shown in FIG. 23 .

Take the day of the tumor inoculation as Day 0. On Day 2, animals wererandomized and administered according to group assignment. Tumors in thevehicle control group grew progressively in all ten mice. Mice wereobserved death on Day 23, and all mice dead on Day 27. The medianduration was 25.5 days.

Compared to vehicle group, mice in CTX group were observed death on Day27, and all mice dead on Day 30. The tumor growth delay (TGD) upontreatment of CTX was 14.74%, with the median duration at 29 days.

Compared to vehicle group, mice in YS-ON-001 group were observed deathon Day ee, and all mice dead on Day 39. The tumor growth delay (TGD)upon treatment of CTX was 38.65%, with the median duration at 34.5 days.

VI. Discussion and Conclusion

In the present example, the vehicle control group had a strong tumorgrowth and the CTX group showed a significant tumor growth delay effectas a positive control.

Treatment with the composition YS-ON-001 at 0.2 mL/mouse showed asignificant tumor growth delay effect, while the animals were welltolerated. The anti-tumor effect of YS-ON-001 is significantly betterthan that of CTX control drug.

The results showed that YS-ON-001 showed strong anti-tumor activity andinhibitory effect on tumor growth in EAC tumor model at the testeddosing level, while the tumor-bearing mice were well tolerated.

Example 9. Therapeutic Effects of the Composition on Thyroid Cancer withLung, Lymph Node Metastasis

1. Subject:

Mao, female, age 53, diagnosed in August 2013 with thyroid cancer withlung metastasis, undergone radical thyroidectomy. Postoperativepathology showed papillary carcinoma on thyroid left lateral lobe, withlymph node metastasis, nodular goiter on thyroid right lateral lobe.Thereafter on Oct. 28, 2013 and Feb. 6, 2014 she undergone twiceiodine-131 therapy. In 2015, she undergone 4 cycles ofEtoposide+Carboplatin radiotherapy (2015.09.28′ 2015.10.20′ 2015.11.07′2015.12.04). After treatment, while 1-2 lesions in lung shrinked, theother 5-6 lesions unchanged, chemotherapy side effects are obvious,physical condition is poor.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With the consent of patient, YS-ON-001 (1 ml) was administered throughintramuscular injection at upper arm, once every 3 days, 12 doses intotal.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects, all lesions are stable, andphysical condition is improved.

Example 10. Therapeutic Effects of the Composition on Kidney Cancer

1. Subject:

Ma, male, age 70, November 2015 began intermittent hematuria symptom,occasional left waist pain. A comprehensive examination was performedand he was diagnosed with left kidney cancer, size about 11×12×6 cm.Chest CT suspected right lung cancer, chronic inflammation of bothlungs, and may be associated with liver cysts. Considered the patient'sage and family opinion, it was decided not to undergo surgery orradiotherapy and chemotherapy. He was discharged after simpleanti-inflammatory treatment.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With the consent of patient and his family, YS-ON-001 (2 ml) wasadministered through intramuscular injection at upper arm from 25thDecember, once every 3 days, 12 doses in total.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects, hematuria symptom and waistpain disappeared.

Example 11. Therapeutic Effects of the Composition on GastricAdenocarcinoma with Metastatic Liver Cancer

1. Subject:

Nan, female, age 55, diagnosed in October 2015 with gastricadenocarcinoma stage IV with metastatic liver cancer, undergoneOxaliplatin+Lapatinib chemotherapy. However the chemotherapydiscontinued due to significant liver damage. After receivingtransaminase reduction treatment and symptomatic treatment, liverfunction basically back to normal.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

3.1 First course of treatment:

With the consent of patient, YS-ON-001 (1 ml) was administered throughintramuscular injection at upper arm from 25 Dec. 2015, once every 3days. This course was discontinued due to abnormal transaminaseelevation on 29th December.

3.2 Second course of treatment:

2 Jan. 2016 liver function recovered till pre-injection level. Secondcourse of YS-ON-001 treatment resumed from 11th January, dosage remainedthe same. Reviewed on 27th January.

4. Results:

After first course of treatment, transaminase abnormally elevated,suspected liver tissue necrosis caused by the administration of thecomposition, believed to be non-specific anti-tumor response.

After second course of treatment, patient showed no obvious sideeffects. Liver CT lesions showed reduced nodal shadow, proofing theconjecture of first course treatment.

Example 12. Therapeutic Effects of the Composition on Breast Cancer withLymphatic, Lung and Osseous Metastasis

1. Subject:

Female, age 34. diagnosed in May 2008 with left breast infiltratingductal carcinoma, undergone modified mastectomy. Case suggested lymphnode metastases, immunohistochemical results showed ER−, PR++, HER2++.June 2015 confirmed both lungs and osseous metastasis. Cranial CT showedmultiple metastases at bilateral cerebellar hemisphere and cerebellarvermis. The condition was judged as disease progressive disease (PD).

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

3.1 First course of treatment:

With consent of patient, from 29 Sep. 2015 onwards YS-ON-001 wasadministered through intramuscular injection, one dose per week (1st,3rd, 5th dose at 2 ml, 2nd, 4th, 6th dose at 1 ml), undergonechemotherapy on doctor's advice.

3.2 Second course of treatment:

From 14th December onwards second course of YS-ON-001 is administeredthrough intramuscular injection, one dose (1 ml) every 3 days.

4. Results:

After first course of treatment, review by chest CT found right axillarylymph nodes swollen, pleural effusion was significantly reduced, rightlung tissues recruited, necrosis on both lung's tissues, brainmetastases disappeared. After a week, disease determined to be stabledisease (SD).

After second course of treatment, chest CT review found both pulmonarynodular shadow obviously decreased. FIG. 24A shows the CT before usingYS-ON-001, and FIG. 24B shows the CT after two courses of treatmentYS-ON-001. Pulmonary nodular shadow was significantly reduced bycomparing the two. The composition of present disclosure has significantantitumor effects.

Example 13. Therapeutic Effects of the Composition on Tongue Cancer

1. Subject:

Zhou, female, age 78, diagnosed with tongue cancer in April 2015,undergone left tongue cancer extended excision+mandibular partialresection+tooth extraction. Postoperative pathology showedwell-differentiated squamous cell carcinoma. In May 2016 left tonguecancer relapsed, undergone extended resection on left tongue recurrentcarcinoma.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With consent of patient, from 26 Jul. 2016 onwards YS-ON-001 wasadministered through buttocks intramuscular injection, 2 doses daily.After administered for 2 days, increased dosage to 4 times daily.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects. Patient declared that upperjaw ulcers depth shallowed, physical condition improved, better sleep,increased appetite, and physical strength was getting better.

Example 14. Therapeutic Effects of the Composition on Prostate Cancerwith Multiple Bone Metastases

1. Subject:

Xu, male, age 81, diagnosed with prostate cancer with multiple bonemetastases in January 2013.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With consent of patient, from 3 Jul. 2016 onwards YS-ON-001 wasadministered through intramuscular injection on every other day, 2 doseseach time. From 3 Aug. 2016, doses increased to 3. From 5 Aug. 2016,injection every other day, each time 4 doses. At the same time Zoladexwas used as combined treatment on doctor's advice.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects, except from muscularstiffness due to long term injection. During treatment, total prostatespecific antigen level reduced, improved physical condition, painrelieved. Before treatment, prostatic puncture biopsy result showed thatall 10 needles tested positive and this number reduced to 5 positive outof 10 after the treatment.

Example 15. Therapeutic Effects of the Composition on Rectal Cancer

1. Subject:

Sun, male, age 63 diagnosed in, November 2012 with rectal cancer,underwent rectal cancer resection+liver biopsy. Postoperative pathologyshowed moderately differentiated rectal ulcer cancer, infiltrated theentire intestine wall, multiple cancer nodules seen inside mesentericadipose tissue, visible metastatic cancer at lymph nodes, metastaticnodules seen in liver. After recovered from operation, from May 2012onwards undergone treatment by taking Bevacizumab (600 mg) and Xelox(OXA 250 mg, Xelod3.0, 14d). After 8 cycles, taking only Bevacizumabtill September 2015. During March 2016 review CT image diagnosisreported multiple intrahepatic metastases, multiple bone metastases overwhole body, left lung lower lobe small nodule metastasis pending forconfirmation.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With consent of patient, from 25 Mar. 2016 onwards YS-ON-001 wasadministered through intramuscular injection. In the first month, 1 dose(1 ml) was administered every other day. On second month, 1 doseadministered each day. On third month, 2 doses daily. From 10th August,3 doses daily.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects. During treatment, patient'ssymptoms unchanged, disease was stable without further complication.Lower extremity edema caused by the use of Bevacizumab was improved.

Example 16. Therapeutic Effects of the Composition on Endometrial Cancer

1. Subject:

Zhang, female, age 56, diagnosed in December 2010 with third stageendometrial cancer, underwent extensive abdominal hysterectomy+radicalovarian cancer surgery. Postoperative pathology found endometrialadenocarcinoma (histologic tumor grade 1), bilateral ovarian cancerinfiltration, right uterosacral ligament metastasis. After surgery,January 2011 onwards started treatment with Docetaxel+Cisplatinchemotherapy. In January 2015 discovered liver and rectal recurrence andmetastases, chemotherapy and cancer pain treatment followed.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration Regimen:

With consent of patient, from 10 Jun. 2016 onwards YS-ON-001 wasadministered through intramuscular injection. For first 10 days 2 doses(2 ml) daily, subsequently 4 doses (4 ml) daily.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects, except from muscularstiffness at injection site. After 2 months treatment, compared toprevious month CT examination, lower density lumps on left abdomen wallslightly reduced, obvious irregular thickening on splenic flexure inproximity, slightly better than before.

Example 17. Therapeutic Effects of the Composition on Ovarian Cancer

1. Subject:

Zhou, female, age 56, diagnosed in June 2016 with stage IIIc ovariancancer (high-grade serous carcinoma), underwent laparotomy for ovariancancer cytoreductive surgery+rectal cancer resection. Postoperativepathology report (left uterine appendages, right ovary) matchedhigh-grade serous carcinoma, rectal lesions, cancerous tissues found atappendiceal serosa and abdominal wall.

2. Composition:

When administered to human subject, YS-ON-001 is formulated in sterilewater for injection.

3. Administration regimen:

With consent of patient, from 16 Jun. 2016 onwards YS-ON-001 wasadministered through intramuscular injection. 2 doses (2 ml) daily forfirst 7 days, followed by 4 doses (4 ml) daily thereafter. At the sametime, Docetaxel and Carboplatin was used as combined treatment ondoctor's advice.

4. Results:

In this example, subject administered with the composition of presentdisclosure showed no obvious side effects. During treatment, patientsymptoms remained unchanged, stable without further progression.

REFERENCES

Any listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat such document is part of the state of the art or is common generalknowledge.

The following references may be of interest:

-   Wanqing Chen, et al. Cancer Statistics in China, 2015. Cancer    Journal for Clinicians. 2016 Vol. 66: 115-132.-   Guoqian Kuang, et al. Current Status and Prospect of Cancer    Virotherapy Clinical Studies. Journal of Guangxi Medical University,    1995 Vol. 12: 617-619.-   Shounan Tan, Fengyu Zhang. Studies on Rabies and Rabies Vaccine for    Human Use. Medical Information 2011 Vol. 24: 2841-2842.-   Yuhui Zhang. The Establishment of Rabies Vaccine Purification    Technology. Chinese Journal of Biologicals 1999 Vol. 12 Iss. No. 4:    231-232.-   Jiang Zhong. Oncolytic Virus and Tumor Treatment, Foreign Medicine    (Microbiology Section), 2004 Vol. 27 Iss. No. 6.-   Kenney S, et al. Viruses as oncolytic agents: a new age for    therapeutic virus. J Nati Cancer Inst, 1994, 86: 1185.-   Lorence, et al. Complete regression of human neuroblastoma    xenografts in athymic mice after local Newcastle disease virus    therapy. J Nati Cancer Inst, 1994, 86: 1228.-   CN100341571C-   RU2414238C2-   US2010/0297072A1-   WO2009/016433

1.-28. (canceled)
 29. A method for treating cancer in a subject,comprising administering to the subject a composition comprising: a)polyinosinic-polycytidylic acid (PIC), b) an antibiotic or a polyaminecompound, c) a positive ion, and d) a virus; wherein the virus isincapable of replication in a human subject.
 30. The method of claim 29,wherein the virus belongs to a family selected from the group consistingof rhabdoviridae, adeniviridae, arenaviridae, astroviridae,bunyaviridae, cliciviridae, flaviviridae, hepatitis delta virus,hepeviridae, mononegavi rales, nidovirales, piconaviridae,orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae,poxviridae, reoviridae, retroviridae, and togaviridae.
 31. The method ofclaim 30, wherein the virus belongs to Lyssavirus genus of rhabdoviridaefamily.
 32. The method of claim 31, wherein the virus is a rabies virus.33. The method of claim 29, wherein the antibiotic is selected from thegroup consisting of kanamycin, tacrolamycin, anthracycline, butyrinsulphate, gentamicin, hygromycin, amikacin, nebramycin, β-lactam,neomycin, puromycin, streptomycin, streptozocin, and any combinationthereof.
 34. The method of claim 29, wherein the composition comprises apolyamine compound, wherein the polyamine compound is selected from thegroup consisting of arginine salt, spermidine, N-(3-aminopropyl),N-(3-aminopropyl)-1,4-butanediamine, spermine,OS-dimethylaminothiophosphate, poly-lysine, aminoglycoside, and anycombination thereof.
 35. The method of claim 29, wherein said positiveion is a cation and is selected from the group consisting of calcium,cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt,deuterium, gallium, iodine, iron, zinc, and any combination thereof. 36.The method of claim 29, wherein cancer comprises lung cancer, breastcancer, thyroid cancer, kidney cancer, gastric adenocarcinoma, livercancer, melanoma, tongue cancer, colon cancer, rectal cancer,endometrial cancer, pancreatic cancer, prostate cancer, cervical cancer,and ovarian cancer.
 37. The method of claim 29, wherein cancer is ametastatic tumor.
 38. The method of claim 29, wherein the cancer is notcaused by the virus.
 39. The method of claim 29, wherein the route ofsaid administration is selected from the group consisting ofintramuscular, intraperitoneal, intravenous, subcutaneous, transdermal,intradermal, intranasal, intraocular, oral, sublingual, intratumoral,and peritumoral.
 40. The method of claim 29, further comprising treatingthe subject with an anti-tumor treatment regimen is selected from thegroup consisting of chemotherapy, radiotherapy, targeted therapy, andimmunotherapy.
 41. The method of claim 29, wherein the ratio of thevirus to the PIC is selected from the group consisting of: 1 IU/50 μg, 1IU/60 μg, 1 IU 70 μg, 1 IU/80 μg, 1 IU/90 μg, 1 IU/100 μg, 1 IU/125 μg,1 IU/200 μg, 1 IU/250 μg, 1 IU/300 μg, 1 IU/350 μg, I IU/400 μg, 1IU/450 μg, 1 IU/500 μg, 1 IU/550 μg, 1 IU/600 μg, 1 IU/700 μg, 1 IU/800μg, 1 IU/1000 μg, 1 IU/1500 μg, 1 IU/2000 μg, 1 IU/2500 μg, 1 IU/3000μg, I IU/4000 μg, 1 IU/5000 μg, 1 IU/6000 μg, 1 IU/7000 μg, 1 IU/8000μg, 1 IU/9000 μg, 1 IU/10,000 μg, and a range between any two of theabove ratios.
 42. The method of claim 29, wherein the ratio of the virusto the PIC 1 IU/500 μg.
 43. The method of claim 29, wherein amount ofthe PIC is between from 250 μg to 5000 μg per unit dose.
 44. The methodof claim 29, wherein amount of the PIC is selected from the groupconsisting of 250 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg, 3000 μg, 4000μg, and 5000 μg per unit dose.
 45. The method of claim 29, wherein theamount of the virus is selected from the group consisting of 0.5 IU, 1.0IU, 1.5 IU, 2.0 IU, 2.5 IU, 3.0 IU, 3.5 IU, 4.0 IU, 5.0 IU, 6.0 IU, 7.0IU, 8.0 IU, 9.0 IU, 10.0 IU, 15.0 IU, 20.0 IU, 30.0 IU, 40.0 IU, 50.0IU, 60.0 IU, 70.0 IU, 80.0 IU, 90.0 IU, 100.0 IU per unit dose, and arange between any two of the above amounts.
 46. The method of claim 29,wherein the amount of the virus is between from 1.0 IU to 5.0 IU perunit dose.
 47. The method of claim 46, wherein the unit dose is preparedinto a volume which is selected from the group consisting of 0.1 ml,0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml,5.0 ml, 10.0 ml, 20.0 ml, 30.0 ml, 40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml,80.0 ml, 90.0 ml, 100.0 ml, 150.0 ml, 200.0 ml, 250.0 ml, and a rangebetween any two of the above volumes.